The present invention relates to a composition. In particular the present invention relates to a pharmaceutical compositionxe2x80x94and to a class of compounds particularly useful in or as said composition.
Cancer remains a major cause of mortality in most Western countries. So far, evidence suggests that oestrogens are the major mitogens involved in promoting the growth of tumours in endocrine-dependent tissues, such as the breast and endometrium. Although plasma oestrogen concentrations are similar in women with or without breast cancer, breast tumour oestrone and oestradiol levels are significantly higher than in normal breast tissue or blood. In situ synthesis of oestrogen is thought to make an important contribution to the high levels of oestrogens in tumours and therefore specific inhibitors of oestrogen biosynthesis are of potential value for the treatment of endocrine-dependent tumours.
Over the past two decades, there has been considerable interest in the development of inhibitors of the aromatase pathway which converts the androgen precursor androstenedione to oestrone. However, there is now evidence that the oestrone sulphatase (xe2x80x9cE1-STSxe2x80x9d) pathway, i.e. the hydrolysis of oestrone sulphate (xe2x80x9cE1Sxe2x80x9d) to oestrone (xe2x80x9cE1xe2x80x9d), as opposed to the aromatase pathway, is the major source of oestrogen in breast tumours. This theory is supported by a modest reduction of plasma oestrogen concentration in postmenopausal women with breast cancer treated by aromatase inhibitors, such as aminoglutethimide and 4-hydroxyandrostenedione and also by the fact that plasma E1S concentration in these aromatase inhibitor-treated patients remains relatively high. The long half-life of E1S in blood (10-12 h) compared with the unconjugated oestrogens (20 min) and high levels of steroid sulphatase activity in liver and, normal and malignant breast tissues, also lend support to this theory.
Singh et al (1997 J Steroid Biochem Mol Biol 61: 185-192), report that the major source of pro-inflammatory cytokines, such as TNF-xcex1 and IL-6 within breast tumours is not well understood but it is thought that tumour infiltrating macrophages and lymphocytes might play a role.
Singh et al (ibid) report that the release of cytokines, such as IL-6 by tumour cells is also associated with enhanced aromatase activity in breast tissue adjacent to the tumour. Singh et al (ibid) also report that both TNF-xcex1 and IL-6 inhibit the growth of MCF-7 breast cancer cells in vitro. In addition, TNF-xcex1 has an inhibitory effect on aromatase activity measured in cultured MCF-7 breast cancer cells. Apparently, these results contrast with the marked stimulatory effect that TNF-xcex1 has on fibroblasts derived from normal and malignant breast tissues (Macdiarmaid et al 1994 Molec. Cell Endoc. 106: 17-21). In addition, when TNF-xcex1 is combined with IL-6, the inhibitory effect on aromatase activity is enhanced. The synergistic inhibitory effect of IL-6 and TNF-xcex1 on aromatase activity in MCF-7 cells also contrasts to the synergistic stimulatory effect that these cytokines have on oestrone sulphatase and oestradiol dehydrogenase activities in these cells.
Singh et al (ibid) also report that a significant reduction in aromatase activity is observed when conditioned media (CM) from monocytes and lymphocytes of an immunosuppressed kidney transplant patient is added to fibroblast cultures from normal breast cells compared with CM from breast cancer cells. These results suggest that the reduced incidence of breast cancer in immunosuppressed kidney transplant patients could result from reduced cytokine production and thus decreased stimulation of oestrogen synthesis.
Previous studies have also shown that where CM from cultured breast cancer cells stimulates aromatase activity, this CM also stimulates the activities of two main enzymes, that is oestrone sulphatase and oestradiol dehydrogenase which are also involved in breast tumour oestrogen synthesis.
Thus, there appears to be a co-ordinated mechanism for regulating the synthesis of oestrogen within breast tumours that is controlled by cytokines. However, it has been postulated that any in vivo stimulatory effect of cytokines in inhibiting tumour growth may be outweighed by their stimulatory effect on enzyme activity associated with oestrogen synthesis (Duncan and Reed 1995 J Steroid Biochem Molec Biol 55:565-572).
Singh et al (ibid) state that while cytokines such as TNF-xcex1 and IL-6 have been shown to play an important role in regulating the activities of enzymes involved in oestrogen synthesis, it is likely that other cytokines and mediators of the inflammatory response are capable of modulating oestrogen synthesis in normal and malignant breast tissue.
Thus, cancer therapies developed so far have included blocking the action or synthesis of hormones to inhibit the growth of hormone-dependent tumours. However, more aggressive chemotherapy is currently employed for the treatment of hormone-independent tumours.
Hence, the development of a pharmaceutical for anti-cancer treatment of hormone dependent and/or hormone independent tumours, yet lacking some or all of the side-effects associated with chemotherapy, would represent a major therapeutic advance.
In fact, Singh et al (ibid) state that xe2x80x9cby understanding the complex mechanisms which govern oestrogen synthesis, it should be possible to devise better preventative and therapeutic strategiesxe2x80x9d against cancersxe2x80x94especially breast cancer.
The present invention seeks to provide a composition suitable for use in the treatment of cancers and, especially, breast cancer.
According to a first aspect of the present invention there is provided a composition comprising i) a compound comprising a sulphamate group (xe2x80x9ca sulphamate compoundxe2x80x9d); and ii) a biological response modifier.
According to a second aspect of the present invention there is provided the use of a composition according to the present invention in the manufacture of a medicament to prevent and/or inhibit tumour growth.
According to a third aspect of the present invention there is provided the use of a composition according to the present invention in the manufacture of a medicament to do any one or more of: prevent or suppress glucose uptake by a tumour; prevent and/or inhibit tumour angiogeneis; disrupt microtubules; induce apoptosis.
According to a fourth aspect of the present invention there is provided the use of a sulphamate compound comprising a steroidal component and an oxyhydrocarbyl group (xe2x80x9coxyhydrocarbyl steroidal sulpharnate compoundxe2x80x9d) in the manufacture of a medicament to do any one or more of: prevent or suppress glucose uptake by a tumour; prevent and/or inhibit tumour angiogeneis; disrupt microtubules; induce apoptosis.
According to a fifth aspect of the present invention there is provided the composition of the present invention for use in medicine.
According to a sixth aspect of the present invention there is provided a method of treatment comprising administering to a subject in need of treatment a composition according to the present invention.
According to a seventh aspect of the present invention there is provided a method of treatment comprising administering to a subject in need of treatment a composition according to the present invention or an oxyhydrocarbyl steroidal sulphamate compound according to the present invention in order to prevent or suppress glucose uptake by a tumour; and/or prevent and/or inhibit tumour angiogeneis; and/or disrupt microtubules; and/or induce apoptosis.
According to an eighth aspect of the present invention there is provided a kit comprising a part i) containing a compound comprising a sulphamate group (xe2x80x9ca sulphamate compoundxe2x80x9d); and a part ii) containing a biological response modifier. The parts of the kit may be independently held in one or more containersxe2x80x94such as bottles, syringes, plates, wells, blister pack etc.
The present invention is advantageous in that it provides a composition suitable for use in the treatment of cancers and, especially, breast cancer.
In addition, the present invention is advantageous in that it provides a compound that is suitable for use in the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer etc.xe2x80x94especially, breast cancer.
Another advantage of the compositions of the present invention is that they may be more potent in vivo than the sulphamate compounds alone or the biological response modifier alone. Moreover, in some aspects the combination of sulphamate compounds and the biological response modifier is more potent than one would expect from the potency of the compound alone i.e. this is a synergistic relationship between them.
In accordance with the present invention the composition of the present invention may comprise more than one biological response modifier.
The term biological response modifier (xe2x80x9cBRMxe2x80x9d) includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chemotactic, haemolytic and thrombolytic factors, cell surface receptors, ligands, leukocyte adhesion molecules, monoclonal antibodies, preventative and therapeutic vaccines, hormones, extracellular matrix components, fibronectin, etc. BRMs may play a role in modulating the immune and inflammatory response in disorders. Examples of BRMs include: Tumour Necrosis Factor (TNF), granulocyte colony stimulating factor, erythropoietin, insulin-like growth factor (IGF), epidermal growth factor (EGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), interferons (IFNs), interleukins, tissue plasminogen activators, P-, E- or L-Selectins, ICAM-1, VCAM, Selectins, addressins etc.
Preferably, the biological response modifier is a cytokine.
A cytokine is a moleculexe2x80x94often a soluble proteinxe2x80x94that allows immune cells to communicate with each other. These molecules exert their biological functions through specific receptors expressed on the surface of target cells. Binding of the receptors triggers the release of a cascade of biochemical signals which profoundly affect the behaviour of the cell bearing the receptor (Poole, S 1995 TibTech 13: 81-82). Many cytokines and their receptors have been identified at the molecular level (Paul and Sedar 1994, Cell 76: 241-251) and make suitable molecules of therapeutic value as well as therapeutic targets in their own right.
More details on cytokines can be found in Molecular Biology and Biotechnology (Pub. VCH, Ed. Meyers, 1995, pages 202, 203, 394, 390, 475, 790).
Examples of cytokines include: interleukins (IL)xe2x80x94such as IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-19; Tumour Necrosis Factor (TNF)xe2x80x94such as TNF-xcex1; Interferon alpha, beta and gamma; TGF-xcex2.
For the present invention, preferably the cytokine is tumour necrosis factor (TNF).
More preferably the cytokine is TNF-xcex1.
TNF is a cytokine produced by macrophages and lymphocytes which mediates inflammatory and immunopathological responses. TNF has been implicated in the progression of diseases which include but are not limited to immunomodulation disorder, infection, cell proliferation, angiogenesis (neovascularisation), tumour metastasis, apoptosis, sepsis, and endotoxaemia.
The necrotising action of TNF in vivo mainly relates to capillary injury. TNF causes necrosis not only in tumour tissue but also in granulation tissue. It causes morphological changes in growth inhibition of and cytoxicity against cultured vascular endothelial cells (Haranka et al 1987 Ciba Found Symp 131: 140-153).
For the preferred aspect of the present invention, the TNF may be any type of TNFxe2x80x94such as TNF-xcex1, TNF-xcex2, including derivatives or mixtures thereof.
Teachings on TNF may be found in the art such as WO-A-98/08870 and WO-A-98/13348.
The TNF can be prepared chemically or it can be extracted from sources. Preferably, the TNF is prepared by use of recombinant DNA techniques.
With this aspect of the present invention the compositions of the present invention are more potent in vivo than the sulphamate compounds alone or TNF alone. Moreover, in some aspects the combination of sulphamate compounds and TNF is more potent than one would expect from the potency of the compound alone i.e. this is a synergistic relationship between them.
In accordance with the present invention the composition of the present invention may comprise more than one sulphamate compound.
The term xe2x80x9csulphamate compoundxe2x80x9d means a compound comprising at least one sulphamate group.
Preferably, if the sulphamate group on the sulphamate compound were to be replaced with a sulphate group to form a sulphate compound then the sulphate compound would be hydrolysable by a steroid sulphatase enzyme (E.C.3.1.6.2).
Preferably if the sulphamate group on the sulphamate compound were to be replaced with a sulphate group to form a sulphate compound and the sulphate compound were to be incubated with a steroid sulphatase enzyme (E.C.3.1.6.2) at a pH 7.4 and 37xc2x0 C. it would provide a Km value of less than 50 mM.
Preferably if the sulphamate group on the sulphamate compound were to be replaced with a sulphate group to form a sulphate compound and the sulphate compound were to be incubated with a steroid sulphatase enzyme (E.C.3.1.6.2) at a pH 7.4 and 37xc2x0 C. it would provide a Km value of less than 50 xcexcM.
The term xe2x80x9csulphamatexe2x80x9d includes an ester of sulphamic acid, or an ester of an N-substituted derivative of sulphamic acid, or a salt thereof.
Preferably, the sulphamate group of the sulphamate compound has the formula: 
wherein each of R1 and R2 is independently selected from H or a hydrocarbyl group.
The term xe2x80x9chydrocarbyl groupxe2x80x9d as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, a hydrocarbon group, an N-acyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
In one preferred embodiment of the present invention, the hydrocarbyl group is a hydrocarbon group.
Here the term xe2x80x9chydrocarbonxe2x80x9d means any one of an alkyl group, an alkenyl group, an alkynyl group, an acyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Preferably, R1 and R2 are independently selected from H or alkyl, cycloalkyl, alkenyl and aryl, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl, N-acyl, or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R1 and/or R2 is alkyl, the preferred values are those where R1 and R2, are each independently selected from lower alkyl groups containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably R1 and R2 are both methyl. When R1 and/or R2 is aryl, typical values are phenyl and tolyl (xe2x80x94PhCH3; o-, m- or p-). Where R1. and R2 represent cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R1 and R2, typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94 to provide a 5-, 6- or 7- membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl, acyl and aryl we include substituted groups containing as substituents therein one or more groups which do not interfere with the sulphatase inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl. A non-limiting example of a hydrocarbyl group is an acyl group.
In some preferred embodiments, at least one of R1 and R2 is H.
Preferably the sulphamate compound is a cyclic compound. In this regard, the sulphamate compound can be a single ring compound or a polycyclic compound. Here, the term xe2x80x9cpolycyclicxe2x80x9d includes fused and non-fused ring structures including combinations thereof.
Thus, preferably the sulphamate compound is of the formula
Exe2x80x94G
wherein E is a sulphamate group and wherein G is a cyclic group.
The cyclic group may be a single ring or it is a polycylic ring structure.
In one aspect, the cyclic group may contain any one or more of C, H, O, N, P, halogen (including Cl, Br and I), S and P.
At least one of the cyclic groups may be a heterocyclic group (a heterocycle) or a non-heterocyclic group.
At least one of the cyclic groups may be a saturated ring structure or an unsaturated ring structure (such as an aryl group).
Preferably, at least one of the cyclic groups is an aryl ring.
Preferably, the sulphamate group is linked to the aryl ring.
If the cyclic group is polycyclic some or all of the ring components of the sulphamate compound may be fused together or joined via one or more suitable spacer groups.
Thus, in accordance with one aspect of the present invention, preferably the sulphamate compound is a polycyclic compound.
Preferably the polycyclic compound will contain, inclusive of all substituents, no more than 50 about carbon atoms, more usually no more than about 30 to 40 carbon atoms.
The polycyclic compound can comorise at least two ring components, or least three ring components, or least four ring components.
Preferably, the polycyclic compound comprises four ring components.
Preferred polycyclic compounds have a steroidal ring componentxe2x80x94that is to say a cyclopentanophenanthrene skeleton, or bio-isosteres thereof.
As is well known in the art, a classical steroidal ring structure has the generic formula of: 
In the above formula, the rings have been labelled in the conventional manner.
An example of a bio-isostere is when any one or more of rings A, B, C and D is a heterocylic ring and/or when any one or more of rings A, B, C and D has been substituted and/or when any one or more of rings A, B, C and D has been modified; but wherein the bio-isostere in the absence of the sulphamate group has steroidal properties.
In this regard, the structure of a preferred polycyclic compound can be presented as: 
wherein each ring Axe2x80x2, Bxe2x80x2, Cxe2x80x2 and Dxe2x80x2 independently represents a heterocyclic ring or a non-heterocylic ring, which rings may be independently substituted or unsubstituted, saturated or unsaturated.
By way of example, any one or more of rings Axe2x80x2, Bxe2x80x2, Cxe2x80x2 and Dxe2x80x2 may be independently substituted with suitable groupsxe2x80x94such as an alkyl group, an allyl group, an hydroxy group, a halo group, a hydrocarbyl group, an oxyhydrocarbyl group etc.
An example of Dxe2x80x2 is a five or six membered non-heterocyclic ring having at least one substituent.
In one preferred embodiment, the ring Dxe2x80x2 is substituted with a ethinyl group.
If any one of rings Axe2x80x2, Bxe2x80x2, Cxe2x80x2 and Dxe2x80x2 is a heterocyclic ring, then preferably that heterocylic ring comprises a combination of C atoms and at least one N atom and/or at least one O atom. Other heterocyclic atoms may be present in the ring.
Examples of suitable, preferred steroidal nuclei rings Axe2x80x2-Dxe2x80x2 of the compounds of the present invention include rings A-D of oestrone and dehydroepiandrosterone.
Preferred steroidal nuclei rings Axe2x80x2-Dxe2x80x2 of the compounds of the present invention include rings A-D of:
Oestrones and Substituted Oestrones, Viz:
oestrone
2-OH-oestrone
2-alkoxy-oestrone (such as C1-6 alkoxy-oestrone, such as 2-methoxy-oestrone)
4-OH-oestrone
6xcex1-OH-oestrone
7xcex1-OH-oestrone
16xcex1-OH-oestrone
16xcex2-OH-oestrone
Oestradiols and Substituted Oestradiols, Viz:
2-OH-17xcex2-oestradiol
2-alkoxy-17xcex2-oestradiol (such as C1-6 alkoxy-17xcex2-oestradiol, such as 2-methoxy-17xcex2-oestradiol)
4-OH-17xcex2-oestradiol
6xcex1-OH-17xcex2-oestradiol
7xcex1-OH-17xcex2-oestradiol
2-OH-17xcex1-oestradiol
2-alkoxy-17xcex1-oestradiol (such as C1-6 alkoxy-17xcex1-oestradiol, such as 2-methoxy-17xcex1-oestradiol)
4-OH-17xcex1-oestradiol
6xcex1-OH-17xcex1-oestradiol
7xcex1-OH-17xcex1-oestradiol
16xcex1-OH-17xcex1-oestradiol
16xcex1-OH-17xcex2-oestradiol
16xcex2-OH-17xcex1-oestradiol
16xcex2-OH-17xcex2-oestradiol
17xcex1-oestradiol
17xcex2-oestradiol
17xcex1-ethinyl-17xcex2-oestradiol
17xcex2-ethinyl-17xcex1-oestradiol
Oestriols and Substituted Oestriols, Viz:
oestriol
2-OH-oestriol
2-alkoxy-oestriol (such as C1-6 alkoxy-oestriol, such as 2-methoxy-oestriol)
4-OH-oestriol
6xcex1-OH-oestriol
7xcex1-OH-oestriol
Dehydroepiandrosterones and Substituted Dehydroepiandrosterones, Viz:
dehydroepiandrosterones
6xcex1-OH-dehydroepiandrosterone
7xcex1-OH-dehydroepiandrosterone
16xcex1-OH-dehydroepiandrosterone
16xcex2-OH-dehydroepiandrosterone
In general terms the ring system Axe2x80x2Bxe2x80x2Cxe2x80x2Dxe2x80x2 may contain a variety of non-interfering substituents. In particular, the ring system Axe2x80x2Bxe2x80x2Cxe2x80x2Dxe2x80x2 may contain one or more hydroxy, alkyl especially lower (C1-C6) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C1-C6) alkoxy, e.g. methoxy, ethoxy, propoxy etc., aLkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
In an alternative embodiment, the polyclic compound may not contain or be based on a steroid nucleus. In this regard, the polyclic compound may contain or be based on a non-steroidal ring systemxe2x80x94such as diethylstilboestrol, stilboestrol, coumarins, and other ring systems. Other suitable non-steroidal compounds for use in or as the composition of the present invention may be found in U.S. Pat. No. 5,567,831.
In formula (I), the at least one sulphamate group is attached to any one or more of the ring components.
Preferably, the polycyclic compound has a steroidal structure and wherein the sulphamate group is attached to the A ring.
Preferably, the sulphamate group is attached to the 3 position of the A ring.
Preferably the sulphamate compound comprises at least one oxyhydrocarbyl group.
A preferred sulphamate compound is an oxyhydrocarbyl steroidal sulphamate compound (i.e. a sulphamate compound comprising a steroidal component and an oxyhydrocarbyl group).
In one embodiment, preferably, the sulphamate compound is an oxyhydrocarbyl steroidal sulphamate compound wherein the sulphamate group is in the 3 position on the steroidal component and/or the oxyhydrocarbyl group is in the 2-position position on the steroidal component.
In one embodiment, preferably, the sulphamate compound is an oxyhydrocarbyl derivative of oestrone sulphamate.
In one embodiment, preferably, the sulphamate compound is an oxyhydrocarbyl derivative of oestrone-3-O-sulphamate.
In one embodiment, preferably, the sulphamate compound is a C1-6 (such as a C1-3) alkoxy derivative of oestrone-3-O-sulphamate.
In one embodiment, preferably, the sulphamate compound is a 2-C1-6 (such as a C1-3) alkoxy derivative of oestrone-3-O-sulphamate.
In one embodiment, preferably, the sulphamate compound is 2-methoxyoestrone-3-O-sulphamate.
The term xe2x80x9coxyhydrocarbyl groupxe2x80x9d as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group.
Thus, the oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.
In one preferred embodiment of the present invention, the oxyhydrocarbyl group is a oxyhydrocarbon group.
Here the term xe2x80x9coxyhydrocarbonxe2x80x9d means any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Preferably the oxyhydrocarbyl group is of the formula C1-6O (such as a C1-3O).
If the sulphamate compound comprises a steroidal nucleus, preferably the A ring has an oxyhydrocarbyl group at the 2 position.
More preferably the group C1-6O is attached to the 2 position of the A ring of a steroidal nucleus.
Preferably, the oxyhydrocarbyl group is an alkoxy.
The alkyl group of the alkoxy substituent is preferably a lower alkyl group containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably, the alkyl group is methyl.
Thus, in a preferred embodiment, if the sulphamate compound comprises a steroidal nucleus the A ring has an methoxy substituent at the 2 position.
Preferably the sulphamate compound is suitable for use as an inhibitor of oestrone sulphatase (E.C. 3.1.6.2).
In one preferred embodiment of the present invention, preferably the sulphamate compound is non-oestrogenic. The term xe2x80x9cnon-oestrogenicxe2x80x9d means exhibiting no or substantially no oestrogenic activity.
In one preferred embodiment of the present invention, preferably the sulphamate compound are not capable of being metabolised to compounds which display or induce hormonal activity.
In one preferred embodiment of the present invention, preferably the composition of the present invention is orally active.
The present invention is based on the highly surprising finding that the combination of a sulphamate compound and a biological response modifier provides an effective treatment of cancer.
More in particular, we have surprisingly found that the composition of the present inventionxe2x80x94and 2-methoxyoestrone-3-O-sulphamatexe2x80x94can prevent or suppress glucose uptake by a tumour and/or prevent and/or inhibit tumour angiogeneis and/or disrupt microtubules and/or induce apoptosis.
In this respect, microtubules, together with microfilaments and intermediate filaments form part of the cytoskeletal system of a cell. Microtubules are responsible for many of cell movements-examples include the beating of cilia and flagella and the transport of membrane vesicles in the cytoplasm. All these movements result from the polymerisation and depolymerisation of microtubules or the actions of the microtubule motor proteins dynein and kinesins. Some other cell movements , such as the alignment and separation of chromosomes during meiosis and mitosis result from both mechanisms. Microtubules also direct cell movement but in some cases, microtubules serve purely structural functions.
A microtubule is composed of subunits that are heterodimers of xcex1-tubulin and xcex2-tubulin monomers. There are two populations of microtubules: stable, long-lived microtubules and dynamic, short lived microtubules. Dynamic microtubules are found when the microtubule structures need to assemble and dissemble quickly. For example, during mitosis, the cytosolic microtubule network characteristic of interphase cells disappears and the tubulin from it is used to form the spindle apparatus which partitions chromosomes equally to the daughter cells. When mitosis is complete, the spindle disassembles and the interphase microtubule network reforms.
Drugs that inhibit mitosis provide a useful means to manipulate the microtubules in a cell. Three drugs: colchicine, vinblastine and taxolxe2x80x94all purified from plantsxe2x80x94have proved to be very powerful probes of microtubule function partly because they bind only to tubulin or microtubules and not to other proteins and also because their concentrations in cells can be easily controlled.
Because of their effects on mitosis, microtubule inhibitors have been widely used to treat illness and more recently as anticancer agents, since blockage of spindle formation will preferentially inhibit rapidly dividing cells like cancer cells. A highly effective anti-ovarian cancer agent is taxol. In ovarian cancer cells, which undergo rapid cell divisions, mitosis is blocked by taxol treatment while other functions carried out by intact microtubules are not affected. A comprehensive review of microtubules can be found in xe2x80x9cMolecular Cell Biologyxe2x80x9d (Ed: Lodish et al 1995 WH Freeman and Co. New York pp 1051-1122).
Apoptosis is induced by MT-targeting drugs, a process which may involve the phosphorylation (and inactivation) of the apoptosis regulator, the bcl-2 protein (Halder, Cancer Res. 57: 229, 1997).
Preferably the composition of the present invention further comprises a pharmaceutically acceptable carrier, diluent, or excipient.
For pharmaceutical administration, the composition of the present invention can be formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc.xe2x80x94such as those for parenteral administration. Approximate effective dose rates are in the range 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average (70 Kg) bodyweight. More usual dosage rates for the preferred and more active compositions will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day. They may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days. For oral administration they may be formulated in tablets, capsules, solution or suspension containing from 100 to 500 mg of composition per unit dose. Alternatively and preferably the compositions will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg. Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the bodyweight of the patient, such variations being within the skill and judgement of the physician.
The composition or compound of the present invention may be administered in any suitable mannerxe2x80x94such as any one or more of oral administration, topical administration (such as by means of a patch), parenteral administration, rectal administration or by inhalation spray.
In the method of treatment, the subject is preferably a mammal, more preferably a human. For some applications, preferably the human is a woman.
For particular applications, it is envisaged that the compositions of the present invention may be used in combination therapies, either with another sulphatase inhibitor, or, for example, in combination with an aromatase inhibitor, such as for example, 4-hydroxyandrostenedione (4-OHA).
In accordance with the present invention, the components of the composition can be added in admixture, simultaneously or sequentially. Furthermore, in accordance with the present invention it may be possible to form at least a part of the composition in sit (such as in vivo) by inducing the expression ofxe2x80x94or increasing the expression ofxe2x80x94one of the components. For example, it may be possible to induce the expression ofxe2x80x94or increase the expression ofxe2x80x94the biological response modifier, such as TNF. By way of example, it may be possible to induce the expression ofxe2x80x94or increase the expression ofxe2x80x94TNF by adding bacterial lipopolysaccharide (LPS) and muramyl dipeptide (MDP). In this regard, bacterial LPS and MDP in combination can stimulate TNF production from murine spleen cells in vitro and tumour regression in vivo (Fuks et al Biull Eksp Biol Med 1987 104: 497-499).
In addition, the present invention contemplates the composition of the present invention further comprising an inducer of the biological response modifierxe2x80x94such as in vivo inducer of the biological response modifier.
The present invention also contemplates the combination of an oxyhydrocarbyl steroidal sulphamate compound according to the present invention (such as 2-methoxyoestrone-3-O-sulphamate) with an inducer of a biological response modifierxe2x80x94such as an in vivo inducer of an in situ biological response modifier.
Examples of suitable sulphamate compounds for use in or as the composition of the present invention, or examples of suitable compounds that can be converted to suitable sulphamate compounds for use in or as the composition of the present invention, can be found in the artxe2x80x94such as PCT/GB92/01587, PCT/GB97/03352, PCT/GB97/00444, GB9725749.7, GB9725750.5, U.S. Pat. No. 5567831, U.S. Pat. No. 5677292, U.S. Pat. No. 5567831, WO-A-96/05216, and WO-A-96/05217, U.S. Pat. Nos. 6,083,978, 6,017,904, 6,011,024, 5,861,390, 5,830,886, 5,616,574, and 5,604,215, and U.S. applications Ser. Nos. 09/638,315, 09/638,314, 09/125,255, 09/319,213, 09/561,453, 09/572,246 and 09/572,237; and each of the foregoing patents and applications, and all documents cited or referenced in each of the foregoing patents and applications including during any prosecution (xe2x80x9cappln. cited documentsxe2x80x99) and all documents referenced or cited in the appln. cited documents, are hereby incorporated herein by reference.
By way of example, PCT/GB92/01587 teaches novel steroid sulphatase inhibitors and pharmaceutical compositions containing them for use in the treatment of oestrone dependent tumours, especially breast cancer. These steroid sulphatase inhibitors are sulphamate esters. Examples of such inhibitors are sulphamate ester derivatives of steroids.
A compound suitable for use in the present inventionxe2x80x94which is also a preferred compound of PCT/GB92/01587xe2x80x94is oestrone-3-sulphamate (otherwise known as xe2x80x9cEMATExe2x80x9d), which has the following structure: 
It is known that EMATE is a potent E1-STS inhibitor as it displays more than 99% inhibition of E1-STS activity in intact MCF-7 cells at 0.1 xcexcM. EMATE also inhibits the E1-STS enzyme in a timexe2x80x94and concentration-dependent manner, indicating that it acts as an active site-directed inactivator.
Preferably, the A ring has a substituent that is an oxyhydrocarbyl group.
Another compound suitable for use in the present invention has at least the following skeletal structure: 
wherein R denotes a sulphamate group as described above.
Preferably, R is the above-mentioned preferred formula for the sulpharnate group. In this regard, it is preferred that at least one of R1 and R2 is H.
Preferably, the A ring has a substituent that is an oxyhydrocarbyl group.
Another compound suitable for use in the present invention has at least the following skeletal structure: 
wherein R denotes a sulphamate group as described above.
Preferably, R is the above-mentioned preferred formula for the sulphamate group. In this regard, it is preferred that at least one of R1 and R2 is H.
Preferably, the A ring has a substituent that is an oxyhydrocarbyl group.
In accordance with a preferred aspect of the present invention, if the sulphamate group of the compound were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activityxe2x80x94i.e. when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37xc2x0 C.
In one preferred embodiment, if the sulphamate group of the compound were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value of less than 50 m Moles when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37xc2x0 C.
In another preferred embodiment, if the sulphamate group of the compound were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value of less than 50 xcexcMoles when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37xc2x0 C.
In a further aspect the present invention provides use of a sulphamate compound for the manufacture of a medicament to prevent and/or inhibit tumour growth; wherein the sulphamate compound is suitable for use as an inhibitor of oestrone sulphatase (E.C. 3.1.6.2); wherein the compound is a polycyclic compound having a steroidal structure, or a bio-isostere thereof; wherein the polycyclic compound comprises at least one sulphamate group attached to the A ring; and wherein the polycyclic compound comprises at least one oxyhydrocarbyl group attached to the A ring.
We have found that sulphamate compounds having an oxyhydrocarbyl substituent on the A ring are potent (and in some cases highly potent) in (i) preventing and/or inhibiting glucose uptake of a tumour and/or (ii) preventing and/or inhibiting tumour angiogeneis and/or (iii) disrupting microtubules and/or iv) inducing apoptosis.
Thus in a further aspect the present invention provides use of a sulphamate compound for the manufacture of a medicament to prevent and/or inhibit glucose uptake of a tumour and/or to prevent and/or inhibit tumour angiogeneis and/or to disrupt microtubules and/or induce apoptosis; wherein the sulphamate compound is suitable for use as an inhibitor of oestrone sulphatase (E.C. 3.1.6.2); wherein the compound is a polycyclic compound having a steroidal structure or a bio-isostere thereof; wherein the polycyclic compound comprises at least one sulphamate group attached to the A ring; and wherein the polycyclic compound comprises at least one oxyhydrocarbyl group attached to the A ring.
A preferred sulphamate compound of the present invention has the formula: 
wherein X is an oxyhydrocarbyl group; and Y is a sulphamate group; and
wherein rings A, B, C and D are independently optionally substituted.
Preferably Y is in the 3-position.
Preferably X is in the 2-position.
For the present invention, preferably the sulphamate compound is an oxyhydrocarbyl steroidal sulphamate compound, in particular 2-methoxyoestrone-3-O-sulphamate, or a pharmaceutically active salt thereof, including analogues thereof.
2-methoxyoestrone-3-O-sulphamate is an analogue of EMATExe2x80x94and can be called 2-methoxy EMATE.
2-methoxy EMATE is the sulphamoylated derivative of a naturally occurring oestrogen metabolite, 2-methoxyoestrone. This compound is formed in the liver by the hydroxylation of oestrone by a 2-hydroxylase, with subsequent metabolism to the methoxy derivative by catechol oestrogen methyl transferase.
2-methoxy EMATE has the formula presented as formula below: 
2-methoxy EMATE is believed to act in vivo, at least in part, by inhibiting tumour angiogenesis.
Thus, in a highly preferred embodiment the sulphamate compound is an oxyhydrocarbyl steroidal sulphamate compound, in particular 2-methoxyoestrone-3-O-sulphamate (2-methoxy EMATE).
In this regard, we have found that a sulphamate compound having a C1-6 (such as a C1-3) alkoxy substituent at the 2 position of the A ring, in particular 2-methoxy EMATE, is highly potent in preventing and/or inhibiting growth of tumours.
The present invention also provides compositions/compounds which:
cause inhibition of growth of oestrogen receptor positive (ER+) and ER negative (ERxe2x88x92) breast cancer cells in vitro by induction of apoptosis.
cause regression of nitroso-methyl urea (NMU)xe2x80x94induced mammary rumours in intact animals (i.e. not ovariectomised).
inhibit the uptake of glucose in cancer cells, in particular in breast cancer cells and breast tumour-derived fibroblasts.
induce apoptosis.
disrupt microtubules (Mts).
act in vivo by inhibiting angiogenesis.
The sulphamate compounds of the present invention may be prepared by reacting an appropriate alcohol with the appropriate sulfamoyl chloride, R1R2NSO2Cl. Preferred conditions for carrying out the reaction are as follows. Sodium hydride and a sulfamoyl chloride are added to a stirred solution of the alcohol in anhydrous dimethyl formamide at 0xc2x0 C. Subsequently, the reaction is allowed to warm to room temperature whereupon stirring is continued for a further 24 hours. The reaction mixture is poured onto a cold saturated solution of sodium bicarbonate and the resulting aqueous phase is extracted with dichloromethane. The combined organic extracts are dried over anhydrous MgSO4. Filtration followed by solvent evaporation in vacuo and co-evaporated with toluene affords a crude residue which is further purified by flash chromatography. Preferably, the alcohol is derivatised, as appropriate, prior to reaction with the sulfamoyl chloride. Where necessary, functional groups in the alcohol may be protected in known manner and the protecting group or groups removed at the end of the reaction.
In summation, the present invention provides compositions for use in treatment of tumours and pharmaceutical compositions containing them.